Investigating the effects of human Carbonic Anhydrase 1 expression in mammalian cells Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Xiaochen Liu, BSc, MSc January 2016 ABSTRACT Amyotrophic Lateral Sclerosis (ALS) is one of the most common motor neuron diseases with a crude annual incidence rate of ~2 cases per 100,000 in European countries, Japan, United States and Canada. The role of Carbonic Anhydrase 1 (CA1) in ALS pathogenesis is completely unknown. Previous unpublished results from Dr. Jian Liu have shown in the spinal cords of patients with sporadic amyotrophic lateral sclerosis (SALS) there is a significant increased expression of CA1 proteins. The purpose of this study is to examine the effect of CA1 expression in mammalian cells, specifically, whether CA1 expression will affect cellular viability and induce apoptosis. To further understand whether such effect is dependent upon CA1 enzymatic activity, three CA1 mutants (Thr199Val, Glu106Ile and Glu106Gln) were generated using two- step PCR mutagenesis. Also, a fluorescence-based assay using the pH-sensitive fluorophore Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) to measure the anhydrase activity was developed. The assay has been able to circumvent the requirement of the specialized equipment by utilizing a sensitive and fast microplate reader and demonstrated that three - mutants are enzymatically inactive under the physiologically relevant HCO3 dehydration reaction which has not been tested before by others. The data show that transient expression of CA1 in Human Embryonic Kidney 293 (HEK293), African Green Monkey Kidney Fibroblast (COS7) and Human Breast Adenocarcinoma (MCF7) cell lines did not induce significant changes to the cell viability at 36hrs using the Water Soluble Tetrazolium-8 (WST8) assay. Wild-type CA1 significantly reduced cell viability in HEK293 using a virally transduced inducible stable expression system at 96hrs and 144hrs of protein induction whereas out of the two mutants used only Thr199Val induced significant toxicity at 144hrs. Wild-type CA1 has also been found i to protect COS7 cells against doxycycline-induced toxicity at 96hrs and 144hrs of protein induction whereas no protective effect was seen by the mutants. Using flow cytometry analysis the results has shown wild-type CA1 expression significantly increased Caspase-3 activation and its downstream molecule Poly (ADP-Ribose) Polymerase 1 (PARP-1) cleavage at 96hrs whereas Glu106Ile only significantly increased Caspase-3 activation. In conclusion, this study marks the first time where CA1 expression has shown to directly cause significant apoptotic toxicity in HEK293 cells and protect against doxycycline-induced toxicity in COS7 cells. Although the implication of this study in ALS requires further investigation, the results here suggest in healthy cells increased levels of CA1 expression may cause onset of toxicity, whereas when cells undergo stress, increased CA1 expression can be protective to prevent further loss in cell viabilities. Despite numerous previous studies that have examined CA1 as potential diseases marker, these results represent for the first time in understanding the effect of CA1 in mammalian cells. ii ACKNOWLEDGEMENTS First and foremost, I would like to express my gratitude to my supervisors, Dr. Jian Liu and Prof. Samar Hasnain. This thesis would not have been possible without their guidance, suggestions, as well as patience in the past four years. Thank you sincerely for giving me the opportunity to undertake this journey in science. I would like to express sincerest gratitude to Dr. Tatsuhiko Kadowaki for his time and patience during the past four years in helping me during the experimental stages. I am grateful for Dr. Ferdinand Kappes for his invaluable help for providing me the DNA plasmids for setting up the inducible stable cell lines as well as his guidance throughout this process. I am grateful for the help of Dr. Meng Huee Lee for allowing me to conduct experiments using his equipment, his guidance during the experimental stage, as well as lending his valuable reagents and DNA plasmids that were crucial for my experiments. I would like to say thank you to Dr. Minyan Wang for her kindness and allowing me to use her equipment. I would like to express gratitude to Dr. David Ruiz-Carillo, Prof. Zhiliang Lv, and Dr. Hebin Liu for their help and suggestions. I want to say thank you to the technicians Mr. Deyi Lu, Miss. Qiaoli Feng, Mrs. Yan Zhang, Mr. Ziwen Xie, Mr. Zhongkai Huang and Mrs. Sijing Meng for their time and help. I want to thank my PhD colleagues in the lab Miss. Xiaoyang Zhang, Mr. Hao Zhang, Mr. Xiaofeng Dong as well as all the other PhD students for their company and help. iii I would like to express appreciation for the PhD Scholarship provided by XJTLU and the University of Liverpool which allowed me to pursue this PhD. Last but not least, I want to thank my parents, Dr. Bo Liu and Dr. Naixia Wang for their love, care and support over the past 26 years and their support during the PhD process. I would also like to thank Miss. Yiran Qian for her love and encouragements when I needed it the most. iv TABLE OF CONTENTS Title page Abstract i Acknowledgements iii Table of contents v List of figures and table ix Supplementary figure and list of appendices xi Glossary of abbreviations xii Chapter One Theoretical background 1 Part One: Background to ALS and the proteins involved 1 Etiology of ALS 1 ALS genetics 4 Superoxide dismutase 1 (SOD1) 4 TAR-DNA Binding protein (TARDBP) 8 Fused in sarcoma (FUS) 11 Hexanucleotide repeat expansion in C9ORF72 13 Other genes associated with ALS 17 Non-genetic causes of ALS 18 Part Two: Carbonic Anhydrase – its biological function and role in human diseases 20 The biological functions of the carbonic anhydrases (CA) 20 Members of the CA family 22 CA1 gene expression and physiological function 24 The enzymatic mechanism of CA1 27 Role of CA in the nervous system 33 CA1 and human disease 39 Potential role of CA1 in motor neuron degeneration in ALS 40 v References 46 Chapter Two Fluorescence-based assay for measuring CA1 enzyme activity 67 Introduction 67 The catalytic process of CA 67 The importance of the Thr199 and Glu106 residues in CA1 catalysis 68 Generating CA1 inactive mutants – what the studies have shown 72 Measuring CA enzyme activity 74 The manometric method 74 The colorimetric method 75 The electrometric method 76 The spectrophotometric method 77 Aim of the study 81 Materials and methods 83 Mutagenesis to generate site directed CA1 mutants 83 Protein expression and purification 87 pH vs. fluorescence calibration and UV spectra profile for the enzyme assay 88 Fluorescence-based assay for measuring relative CA1 anhydrase activity 89 Results 91 Establishing a modified method for measuring CA1 anhydrase activity 91 The linear relationship between pH and Pyranine fluorescence 94 bCA2 exhibits in vitro activity linearly correlated with the concentration 95 Mutant CA1 proteins do not retain activity in vitro 101 Discussion 106 Development of a modified fluorescence-based assay of measuring CA activities 106 Mutant CA1 are enzymatically inactive in vitro 108 References 115 vi Chapter Three Investigating the effect of CA1 in mammalian cells by transient expression 118 Introduction 118 Effect of CA1 expression on cell survival 118 Measuring CA1-induced cell viability changes 119 Aim of the study 120 Materials and methods 122 Cell culture 122 Transient transfection of human CA1 into mammalian cells 122 Cell viability via WST8 assay 122 Western blot analysis 123 Results 124 Effect of transient CA1 expression in COS7 cell line 124 Effect of transient CA1 expression in HEK293 cell line 125 Effect of transient CA1 expression in MCF7 cell line 126 Discussion 127 Possible explanations for non-significant changes due to CA1 expression 127 Discussion of the study approach 129 References 131 Chapter Four Investigating the effect of CA1 in mammalian cells by induced stable expression 134 Introduction 134 CA isoforms and cell viability 134 CA and optimization of intracellular pH 134 CA1 and cell toxicity 135 Using the lentiviral inducible expression system for studying CA1 136 Materials and methods 138 Cell culture 138 vii Lentiviral stable cell line production 138 Immunofluorescence 140 Western blot analysis 140 Cell viability assay 141 Flow cytometry analysis 141 Results 142 COS7 and HEK293 cells stably express eGFP and CA1 upon induction 142 Doxycycline induced significant toxicity in COS7 cells 146 Wild-type CA1 protein protect COS7 cells from doxycycline induced toxicity 148 Expression of CA1 proteins induces apoptosis in HEK293 cells 150 Discussion 153 Novel discovery of intracellular CA1-induced toxicity in HEK293 cells 153 Wild-type CA1 expression briefly mitigated doxycycline induced toxicity 154 in COS7 cells Different response to CA1 may be due to differences in cell-environment 155 CA1 mutants causing cellular toxicity and its implications 156 References 159 Chapter Five Concluding Remarks: Improved understanding for the role of CA1 162 in human disease and ALS Study objective and experimental approach 162 Brief summary of results presented in previous chapters 163 Possibility of CA1-induced toxicity in HEK293 cells is activity dependent 163 Increased CA1 expression can transiently mitigate doxycycline-induced stress 166 Summary 168 References 170 viii LIST OF FIGURES AND TABLE Page no. Figure 1.1. Protein sequence alignment between CA1 and CA2. 27 Figure 1.2. Schematic drawing of the CA1 protein. 28 Figure 1.3. Zinc-bound histidines and water in the CA1 active site. 29 Figure 1.4. Equations showing the principles of the CA reversible hydration of CO2 30 Figure 1.5. The four histidines assisting proton transfer in CA1. 31 Figure 1.6. Thr199 and Glu106 residues in CA1. 32 Figure 1.7. Protein sequence alignment between human CA3 to CA1 and CA2.